Adipose microenvironment promotes hypersialylation of ovarian cancer cells

Introduction Ovarian and other peritoneal cancers have a strong tendency to metastasize into the surrounding adipose tissue. This study describes an effect of the adipose microenvironment on upregulation of sialic acid-containing glycans in ovarian cancer (OC). Heterogeneous populations of glycosylated OC tumors converged to a highly sialylated cell state that regulates tumorigenesis in an immune-dependent manner. Methods We modeled the adipose microenvironment by conditioning growth media with human patient-derived adipose tissue. OC cell lines grown in the presence vs. absence of adipose conditioned media (ACM) were characterized by transcriptomics, western blotting, and chemical biology glycan labeling methods. Fluorescence-activated cell sorting was used to separate adipose-driven upregulation of hypersialylated (“SNA-high”) vs. hyposialylated (“SNA-low”) OC subpopulations. The two subpopulations were characterized by further transcriptomic and quantitative polymerase chain reaction analyses, then injected into a syngeneic mouse model. Immune system involvement was implicated using wild type and athymic nude mice with a primary endpoint of overall survival. Results Adipose conditioning resulted in upregulation of sialyltransferases ST3GAL1, ST6GAL1, ST6GALNAC3, and ST8Sia1. In culture, OC cells displayed two distinct sialylated subpopulations that were stable for up to 9 passages, suggesting inherent heterogeneity in sialylation that is maintained throughout cell division and media changes. OC tumors that implanted in the omental adipose tissue exclusively reprogrammed to the highly sialylated subpopulation. In wild type C57BL/6 mice, only the hypersialylated SNA-high subpopulation implanted in the adipose, whereas the hyposialylated SNA-low subpopulation failed to be tumorigenic (p=0.023, n=5). In the single case where SNA-low established a tumor, post-mortem analysis revealed reprogramming of the tumor to the SNA-high state in vivo. In athymic nude mice, both subpopulations rapidly formed tumors, implicating a role of the adaptive immune system. Conclusions These findings suggest a model of glycan-dependent tumor evolution wherein the adipose microenvironment reprograms OC to a tumorigenic state that resists the adaptive immune system. Mechanistically, adipose factors upregulate sialyltransferases. To our knowledge, this is the first demonstration of the effect of adipose microenvironment on OC tumor sialylation. Our results set the stage for translational applications targeting sialic acid pathways in OC and other peritoneal cancer tumorigenesis and metastasis.


Introduction
Sialylation, the addition of negatively charged sialic acid sugars on terminal ends of glycans, is upregulated in most cancers and implicated across nearly all phases of cancer progression (1).Sialic acids are 9-carbon hexosamine sugars that cap the ends of glycan chains on proteins and lipids, with roles in altered adhesion and invasion, resistance to apoptosis and immune evasion (2)(3)(4).Sialic acids are added to growing glycan chains by twenty different sialyltransferase (STase) enzymes, which fall under one of four groups: ST3GAL and ST6GAL add sialic acid to galactose; while ST6GALNAC and ST8SIA add sialic acid to N-acetylgalactosamine or sialic acid, respectively (5).STases add sialic acids either through an a-2,3 (in the case of ST3GAL), a-2,6 (in the case of ST6GAL and ST6GALNAC), or a-2,8 (in case ST8SIA) linkage, which refers to the stereochemistry and position of sialic acid relative to the preceding sugar residue.Upregulated expression of STases in cancers have been shown to occur through DNA hypomethylation, gene amplification or as a result of oncogene activity (5).Hypersialylation has been correlated with pro-tumor functions in various cancer types (6)(7)(8).A current gap in knowledge is the lack of clear understanding on how the tumor microenvironment regulates cancer cell sialylation (9).
Peritoneal cancers such as pancreas, colon, gastric, and ovarian exhibit strong predilection to adipose rich-niches in the peritoneal cavity (10-13).These sites include the adipose-rich omentum as well as the mesenteric and perigonadal adipose (14).Adipose tissues not only serve as an energy depot that can sustain the energy requirements of rapidly growing cancer cells, but can also exert paracrine and endocrine effects by secreting adipokines, cytokines, and chemokines that can support cancer cell migration and invasion (15).
Ovarian cancer (OC), by mortality rate, is the deadliest of all gynecological cancers (16,17).A key driver of mortality is that OC is often diagnosed at a late, already metastatic, stage (16,17).The adipose-rich omentum is an early and primary site of OC metastasis (18)(19)(20)(21).The chemokine interleukin 8 (IL-8) is secreted by adipocytes and has been shown to chemoattract OC cells very early in the process of metastasis formation (21).Within the adipose niche, cross talk between adipocytes and OC cells leads to metabolic reprogramming in both cell types, which provide OC cells the required energy to sustain rapid cancer growth.Moreover, the adipose microenvironment has been shown to confer chemoresistance through Akt (22) and Bclxl signaling pathways (23).Following treatment, the adipose microenvironment is also a frequent site of residual and recurrent OC (24)(25)(26).The importance of the adipose microenvironment in OC progression is underscored in studies demonstrating that the extent of tumor debulking in the adipose-rich omentum (24).The response of adipose-associated metastatic disease to chemotherapy is directly proportional to patient survival (27).
In this study, we demonstrate that the adipose microenvironment is a critical regulator of OC cell sialylation.Using in vitro and in vivo assays and both human and mouse models of OC.We showed that secrete factors from omental cultures upregulated several STases and hence reprogrammed overall OC cell sialylation.Further, we demonstrate enhanced tumor establishment by hypersialylated OC cells in an immune dependent manner, with different tumor growth kinetics and overall survival changes in immune-competent vs. immune-incompetent animals.Our results demonstrate that adipose-induced sialylation reprogramming has significant clinical implications in the targeting of sialylation as therapy for OC and other peritoneal cancers.

Generation of human adipose conditioned media
Adipose conditioned media (ACM) were prepared as previously described (23,41).Briefly, 0.5 g of omentum tissue was minced with sterile razor blades and cultured in 10 mL DMEM/F12 media supplemented with 1% exosome-depleted fetal bovine serum (System Biosciences, Palo Alto, CA).ACM was collected the following day, centrifuged at 1500 RPM for 5 minutes, and stored at -80°C until use.

RNA sequencing and data analysis
mRNA-seq primed from the polyA was used to determine expression profiles.Lexogen's QuantSeq 3'mRNA-seq Library Prep Kit (FWD for Illumina) was utilized for building RNA-seq libraries from 0.1-200 ng of total RNA in 5 µl of nuclease-free ultrapure water.Libraries were quantified on the Qubit and Agilent 2200 Tapestation using the DNA High Sensitivity Screen tape.The electrophoretogram, RNA Integrity Number (RIN), and the ratio of the 28S:18S RNA bands are collectively examined to determine overall quality of the RNA.The barcoded libraries were multiplexed at equimolar concentrations and sequenced with 75 bp reads on an Illumina NovaSeq SP flow cell.Average sequencing depth was 1.7x10 7 reads per sample.Data was demultiplexed using Illumina's CASAVA 1.8.2 software.After read quality was assessed (42), reads were aligned to the human genome (Build hg38) (43) and tabulated for each gene region (44).Differential gene expression analysis was used to compare transcriptome changes between conditions using a paired design (45).Significantly altered genes (unadjusted p-value ≤ 0.05) were input in iPathwayGuide (Advaita Bioinformatics, Ann Arbor, MI) to identify differentially regulated Pathways.Significantly impacted pathways were those with combined overrepresentation and pathway perturbation with unadjusted p-value < 0.05.Data generated from RNA sequencing is publicly available in Gene Expression Omnibus at GSE269831.

Protein lysis, SDS-PAGE and western blot analysis
Whole cell protein lysates were isolated by resuspending cell pellets in 1x Cell lysis buffer (Cell Signaling Technologies) with added Complete ™ Protease Inhibitor Cocktail (Millipore Sigma), followed by centrifugation for 20 minutes at 13,000 rpm.Protein lysates were quantified using BCA assay.50 mg of protein lysate was electrophoresed on 12% SDS-polyacrylamide gels and transferred to PVDF membranes (EMD Millipore).After blocking with 5% milk, membranes were probed overnight with primary antibodies at 4°C and incubated with an appropriate secondary antibody for 1 hour at room temperature.The blots were developed using enhanced chemiluminescence and imaged using GE ImageQuant LAS 500 chemiluminescence (Cytiva Life Sciences).The following antibodies were used: ST3GAL1 (RRID: AB_3096968) and GAPDH (RRID : AB_1078991).

RNA extraction and RT-qPCR
RNA was extracted using RNeasy kit (Qiagen) following manufacturer's instructions.One mg RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratories) and 1:10 dilution of cDNA was used for each qPCR reaction.qPCR was performed using TaqPath ™ qPCR Master Mix, CG (Thermo Fisher Scientific: A55866) with the following TaqMan primers: St3Gal1 (Thermo Fisher Scientific Assay ID: Mm00501493_m1); St6Gal1 (Thermo Fisher Scientific Assay ID: Mm00486119_m1); St6GalNac3 (Thermo Fisher Scientific Assay ID: Mm01316813_m1); and RPS17 (Thermo Fisher Scientific Assay ID: Mm01314921_g1).qCPR was run on CFX96TM PCR detection system (Bio-Rad) using the following thermocycling parameters: polymerase activation at 95°C for 20 secs followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min.Relative expression was calculated using the comparative DDCT method.No RT samples were used as negative control.All reactions were performed in triplicates.

In vivo studies
All the described experiments using mice were approved by Wayne State University Animal Care and Use Committee (IACUC 22-03-4474) and mice were housed at Wayne State University Division of Laboratory Animal Resources.Mouse OC cells were injected intra-peritoneally (i.p.) in 7 week old female C57BL/6 mice (RRID : IMSR_JAX:000664; Jackson Laboratories, strain 000664) at 1×10 7 or in athymic nude mice (Inotiv (Envigo) Hsd : Athymic nude-Foxn1 nu , strain 6905F) at 5×10 6 .mCherry fluorescence was measured by live imaging under isoflurane anesthesia twice weekly using Ami HT Imaging System (Spectral Instruments).Mice were imaged with an Excitation of 570nm, emission of 630nm.Tumor burden was quantified using mCherry region of interest (ROI) using Aura Imaging Software (Spectral Instruments).mCherry ROI area exceeding 3.4x10 8 photons/sec (for C57BL/6) or 5x10 8 photons/sec (for athymic nude mice) were considered above background based on imaging of non-tumor bearing mice.Animals were sacrificed when mCherry ROI area exceeded 1×10 9 photons/second for two consecutive images or when abdominal width reached or exceeded 3.4 cm.All animals were included in the analysis and investigators were not blinded to groupings.

Statistical analysis
Unpaired two-tailed Student t tests, assuming Gaussian distribution, or one-way or two-way analysis of variance (ANOVA) with multiple comparisons were used for comparison between different groups.Log-rank (Mantel-Cox) test was used for survival analysis.P values of 0.05 or less were considered statistically significant.Statistical analysis was performed, and all data were graphed, using GraphPad Prism v9.3.1 (San Diego, CA; RRID : SCR_002798).Data are presented as mean ± SEM.

Adipose upregulates ovarian cancer cell sialylation
Given the significance of the adipose microenvironment in OC progression (21,22,41,46,47) we set to identify mechanisms induced by chronic exposure of OC cells to adipose secreted factors.Since adipocytes represent the primary cell type in the omentum, we obtained adipose-conditioned media (ACM) from dissociated human omentum (23,41), treated human A2780 OC cells for 7 days with ACM, and performed transcriptomic analysis.Of the 27,162 measured genes, we observed 593 differentially expressed genes (DEGs; p<0.05; fold-changed (FC)>0.6)relative to non-ACM-treated control cells (Figure 1A).Pathway Enrichment and Pathway Impact analyses showed 11 differentially regulated pathways (Table 1, Figure 1B) and one of them was the glycosaminoglycan biosynthesis pathway (p=0.036; Figure 1B, yellow dot).In the glycosaminoglycan biosynthesis pathway, two genes were significantly upregulated in ACM-treated cells: B3GNT7 (p=0.039), which encodes b1-3-N-acetylglucosaminyltransferase and ST3GAL1 (p=0.034), which encodes a STase (Figure 1C).The increase in ST3GAL1, one of several STases that catalyze the addition of sialic acid to terminal ends of glycans, was validated at the protein level using two different patient omenta (Figure 1D; Supplementary Figure 1).These results suggested that the adipose environment can elevate sialylation in OC cells.
To determine if adipose-induced upregulation of STases leads to a measurable increase in cell surface sialylation, we used copperindependent and strain-promoted azide-alkyne click chemistry (SPAAC) to directly label sialic acid sugars.SPAAC reactions connect an azide species and alkyne via a bioorthogonal cycloaddition reaction (48)(49)(50).The azide component was installed on sialic sugars through feeding tetraacetylated Nazidoacetyl-mannosamine (ManNAz), which is metabolized and converted to sialic acid azide in cells and added to terminal ends of sialoglycans.The strained alkyne component we used was dibenzocyclooctyne (DBCO) coupled to AF488 fluorophore (DBCO-AF488).When DBCO-AF488 was added to cells that had metabolically incorporated azides on cell surface sialic acids, the reaction resulted in a covalent bond that linked the fluorophore to cell surface sialic acids (Figure 2A).We first determined basal sialic acid expression.Click chemistry performed on mCherry+ TKO mouse OC cells showed membranal green staining demonstrating cell surface sialylation (Figure 2B).To quantitate the effect of adipose conditioning on sialic acid expression, we treated R182 human OC cells with ACM for 72 h and ManNAz was added during the last 24 h of treatment (Figure 2Ci).At the end of the treatment, cells were incubated with DBCO-AF488.Quantification of fluorophore signal showed significant upregulation of cell surface sialic acid expression with ACM compared to ManNaz only control (Figure 2Cii).These results showed that OC cells expressed basal cell surface sialoglycans, which were significantly enhanced by adipose.animals and are classically used to evaluate glycan structures.SNA preferentially binds a-2,6-linked sialic acids and is a good indicator of ST6GAL1 activity (51).MAL -I and Mal-II preferentially bindà -2,3-linked sialic acids, the enzymatic products of ST3GAL1 (52-54).Finally, PNA detects non-sialylated galactose, which is one of the required precursors to sialic acid modification on cell surface glycans (55).We first characterized basal cell surface sialylation and used these lectins to stain a panel of human (OCSC1-F2, R182, OVCAR3 and OVCA432; Supplementary Figure 2) and mouse (TKO and ID8p53KO; Supplementary Figure 3) OC cell lines.All human cell lines showed positive staining for SNA and PNA, although with varying intensity (Supplementary Figure 2).Human cell lines with higher staining for SNA (i.e.R182 > OCSC1-F2) showed lower staining for PNA, as expected.MAL-I staining was only observed in OVCAR3 and MAL-II staining was observed in R182, OVCAR3 and OVCA432 but not in OCSC1-F2.Sialylation pattern on the two mouse cell lines tested was also variable.Both mouse cell lines showed positive staining for SNA and PNA (Supplementary Figure 3).Only TKO showed positive staining for MAL-II.Neither of the mouse cell lines stained positively for MAL-I.
Having characterized basal cell surface sialylation, we then utilized the OCSC1-F2 human OC cells to determine the effect of adipose on specific sialic acid linkages.Thus, OCSC1-F2 cells were treated for 7 days with ACM obtained from three different patient omenta prior to lectin staining.Compared to control cultures, OCSC1-F2 cells treated with ACM showed a trend of increased cell surface expression of a-2,6and a-2,3-linked sialic acids, as detected by SNA and MAL-I staining, respectively.Despite this trend, statistical significance was not reached (Figures 3A, B), suggesting that the effects had relatively high variability.Minimal increase in MAL-II staining and a decrease in PNA staining were observed in ACM-treated cells but the difference from control cells was also not statistically significant (Figures 3C, D).
We noted in our lectin staining panels that TKO mouse OC cells reproducibly generated two distinct SNA-staining populations in vitro (Supplementary Figure 3).Over time, the percentage of cells in these two sub-populations fluctuated between ca.20-60%, but the two distinct populations were persistent and were maintained in culture when followed until 9 passages (Supplementary Figure 4).Flow cytometry assisted cell sorting (FACS) allowed us to further interrogate sialylation on these two cell subpopulations.After authentication through STR profiling (Supplementary Figure 5), lectin staining comparing TKO SNAhigh and TKO SNAlow cells showed that these cultures were only different in SNA staining for a-2,6linked sialic acids and demonstrated comparable staining for a-2,3linked sialic acids via MAL-I and MAL-II (Supplementary Figure 6).TKO SNAhigh cells showed slightly lower PNA staining compared to TKO SNAlow cells (Supplementary Figure 6).SNA staining remained stable in both TKO SNAhigh and TKO SNAlow cells when followed through different passages (Figure 4B).To further demonstrate the effect of adipose secreted factors, we treated TKO SNAlow cells with ACM.We noted an increase in SNA and MAL-I in ACM-treated TKO SNAlow cells (Figure 5), which parallels what was observed with ACM-treated OCSC1-F2 human OC cells (Figure 3).In TKO SNAlow cells, we also noted a decrease in PNA staining upon treatment with ACM (Figure 5).No changes were observed with MAL-II staining.Taken together, these results demonstrate that adipose secreted factors can upregulate both a-2,6and a-2,3-linked sialic acids in both human and mouse ovarian cancer cells.

In vivo engraftment reprograms ovarian cancer cell sialylation
To determine if the adipose-induced increase in sialylation observed in vitro is recapitulated in vivo, we established i.p tumors from parental mCherry+ TKO mouse OC cells in C57BL/ 6 mice.We previously reported the characterization of i.p. ovarian tumors formed by this model and its preferential seeding to omentum, pelvic fat, and mesenteric adipose (51,56).Necropsy showed omental implants (Figure 6) as previously reported (51,56).We then compared cell surface sialic acid expression between TKO cancer cells in culture (Figure 6, top panel) and dissociated TKO cancer cells from the omentum implants (Figure 6, bottom panel).Interestingly, we observed sialylation reprogramming upon in vivo engraftment.Unlike TKO cells in culture, which showed two peaks for SNA staining, TKO cells from dissociated omental tumors showed a single SNA peak, which matched the staining intensity observed in the TKO SNAhigh cell population (Figure 6).In addition, TKO cells from dissociated tumors showed increase in both MAL-I and MAL-II staining and decrease in PNA compared to TKO cells in culture (Figure 6; Supplementary Figure 7).These results demonstrate that in vivo engraftment may favor or select TKO SNAhigh cells.Additionally, these data demonstrate a broad reprogramming of sialylation in ovarian tumors with increase in both a-2,6and a-2,3-sialic acid linkages.

Heterogeneous pool of sialylated ovarian cancer cells in culture
The finding in TKO mouse OC cultures of two subpopulations of cells based on SNA staining is in line with previous reports of heterogeneity in expression of a-2,6 sialic acids in breast and lung cancer cultures (57)(58)(59).We further confirmed that these cells have differential surface sialic acid levels by treating TKO SNAhigh and TKO SNAlow cells with neuraminidase.Neuraminidase removes all cell surface sialic acids and exposes the underlying galactose, which can be bound by PNA (Supplementary Figure 8).Indeed, baseline Adipose-conditioned media (ACM) upregulate a2,6 and a2,3 sialic acids in mouse OC cells.TKO SNAlow cells were sorted by FACS and treated with ACM24 for 7 days prior to lectin staining.PNA staining showed that TKO SNAhigh cells had lower PNA staining compared to TKO SNAlow cells (Supplementary Figure 8) and thus higher cell surface sialic acid in TKO SNAhigh cells.After neuraminidase treatment however, both cell populations showed comparable PNA staining further proving initial difference in cell surface sialic acid levels between the two cell subpopulations.

Hyposialylated ovarian cancer cells fail to form tumors in an immunedependent manner
The observation that parental TKO OC cells formed i.p. tumors that consisted of only TKO SNAhigh cells (Figure 5) suggested that TKO SNAlow cells are not tumorigenic.To test this hypothesis, we injected each subpopulation (Supplementary Figure 9) i.p. in immune-competent C57BL/6 mice.We observed tumor formation only in mice administered TKO SNAhigh cells.Logarithmic tumor growth was seen in these mice beginning at day 30 (Figures 7A, B).In contrast, mice injected with TKO SNAlow cells demonstrated measurable disease only immediately after injection (day 3), after which point the signal dropped and all but one mouse remained disease-free until day 70 (Figures 7A, B).As such, tumor growth rate was significantly different in TKO SNAhigh group compared to TKO SNAlow group (p<0.0001; Figure 7A) and mice in TKO SNAhigh group showed significantly shorter overall survival (p=0.035; Figure 7C).These results show that SNA/sialic acid enriches for OC cells that are tumorigenic in immunecompetent mice.
The observed difference in tumorigenic potential between TKO SNAhigh and TKO SNAlow cells in immune competent mice may be due to cell-intrinsic mechanisms or these differences may be immune related.Analysis of cell growth in culture showed comparable growth rate for the two cell populations (Supplementary Figure 10).To determine the contribution of the immune system we injected each cell population in athymic nude mice lacking T cells.Interestingly, in the absence of T cells, TKO SNAlow cells were able to form tumors, albeit with slower kinetics (Figures 8A, B).Mice injected TKO SNAlow cells showed significant delay in tumor formation (p=0.04) but there was no significant difference in overall survival (Figures 8A, C).Taken together, our results demonstrate that the tumorigenic capacity of Frontiers in Oncology frontiersin.orghyposialylated OC cells can be fully inhibited by the adaptive immune system.In contrast, the innate immune system can only delay but not fully prevent its tumorigenic potential.

tumor formation by hyposialylated ovarian cancer cells in immune-compromised mice leads to hypersialylation
The observation that TKO SNAlow cells form tumors in immune deficient mice provided a platform to further validate in vivo sialylation reprogramming.Thus, we characterized the tumors formed by both the TKO SNAhigh and TKO SNAlow cells in athymic nude mice.Necropsy showed that majority of the i.p. tumors were seeded in the omentum (Figure 8D).We then established cultures from dissociated omental explants and characterized their sialylation levels.SNA staining showed comparably high SNA intensity between explants from TKO SNAhigh and TKO SNAlow tumors demonstrating that TKO SNAlow cells are re-programmed in vivo to gain a-2,6-sialylation (Figure 8D).In addition to equivalent SNA staining, explants from both groups showed positive staining for Mal-II demonstrating gain in a-2,3sialylation as well (Figure 8D).Finally, we measured the levels of St3Gal1, St6Gal1, and St6GalNac3 in the tumor explants.qPCR data showed significant increase in all STases in the tumor explants compared to TKO SNAlow cells grown in culture (Figure 8E).This data replicates what was found in vitro ACM treatment (Figure 5).In both in vitro ACM treatment (Figure 5) and in vivo tumor implantation (Figure 8D), OC cells showed increased sialylation.Taken together, our results demonstrated that adipose factors reprogrammed OC to a hypersialylated state, and that hypersialylation in OC cells resulted in immune system avoidance and a decrease in OC survival.

Discussion
We demonstrate in this study that the adipose microenvironment is a critical regulator of OC cell sialylation.We first took a broad approach to how secreted factors from adipose-rich omentum impacted the OC cell transcriptome.We discovered a significant effect on the STase, ST3GAL1, and using human and mouse models of OC further characterized adipose-induced sialylation.Upon adipose conditioning in vitro, both human and mouse OC cells increased sialylation for both a-2,3 and a-2,6 linked sialic acids.Further, in vivo engraftment, which for OC typically occurs in the omentum, also lead to increased overall sialylation.Mechanistically, we observed increased expression of not only ST3GAL1, but also ST6GAL1 and ST6GALNAC3 upon in vivo engraftment.Intriguingly, we discovered two distinct subpopulations of OC cells with low or high a-2,6-sialic acid levels.When separately injected into immune-

3. 2
Adipose upregulates a-2,6and a-2,3linked sialic acids on ovarian cancer cells For a more comprehensive characterization of cell surface sialylation, we used a panel of four fluorophore-tagged lectins: Sambucus nigra lectin (SNA), Maackia amurensis Lectin I (MAL I), Maackia amurensis Lectin II (MAL II), and peanut agglutinin (PNA).Lectins are sugar binding proteins isolated from plants and Adipose-conditioned media (ACM) upregulate sialyltransferases in human ovarian cancer cells.A2780 human OC cells were treated with ACM for 7 days prior to RNA sequencing.Control cells were maintained in growth media.(A) Volcano plot of differentially expressed genes (DEGs;p<0.05and fold-change>0.6)comparing Control vs ACM-treated cells; position of ST3GAL1 and B3GNT7 are shown; (B) Differentially regulated pathways showing both Pathway impact (pORA) and Pathway enrichment (pAcc); red dots are differentially regulated and Pathway names are shown in Table 1; yellow dot corresponds to Glycosaminoglycan biosynthesis pathway (p=0.036);(C) DEGs within Glycosaminoglycan pathway: B3GNT7 (p=0.039) and ST3GAL1 (p=0.034);(D) Western blot analysis of human R182 ovarian cancer cells treated with ACM from either patient W or patient Y showing upregulation of ST3GAL1.NT, no treatment control.

2 3
FIGURE 2 Adipose-conditioned media (ACM) upregulate cell surface sialylation in human and mouse ovarian cancer cells.(A) Diagram of click chemistry detailed in text; (B) mCherry+ TKO mouse OC cells were treated with 50 mM ManNAz everyday for 3 days followed by treatment with DBCO-FITC.Microscopy analysis shows basal expression of cell surface sialoglycans; (C) i, treatment protocol with ACM prior to click chemistry; ii, R182 human OC cells were treated as in Ci and mean intensity of AF488 was quantified.Note basal sialylation, which is upregulated by ACM treatment.Data are presented as mean ± SEM (n=3); ** p = 0.0062 by One-Way ANOVA with post-hoc multiple comparison analysis.

FIGURE 6
FIGURE 6    In vivo engraftment upregulates general sialylation.top panel, Cell surface sialic acid expression in mCherry+ TKO mouse OC cells in culture as detected by SNA, MAL-I, MAL-II and PNA; bottom panel, mCherry+ TKO mouse OC cells were injected i.p. in C57BL/6 mice and omental tumors were dissociated, stained with anti-CD45 and FITC-tagged lectins (n=5).Histograms show FITC staining from CD45-negative population.Note loss of SNA-low population and increase in Mal-I and Mal-II upon in vivo tumor formation.Histograms show data from one mouse.Similar results were observed in other mice.Double sided arrow shows SNA levels in tumors is comparable to SNA levels in TKO SNAhigh cells.

7 TKO
FIGURE 7 TKO SNAlow cells do not form tumors in immune-competent mice.1x10 7 TKO SNAhigh or TKO SNAlow cells were injected i.p. in female C57BL/6 mice (n=5).mCherry fluorescence was acquired every 3-4 days and mCherry ROI area was quantified as measure of i.p. tumor burden.(A) Tumor growth curves showing significant difference in measured mCherry ROI between groups (p<0.0001 by Two-Way ANOVA).Dashed line shows threshold for mCherry signal; (B) Representative images obtained from live imaging; (C) Kaplan-Meir survival curve (Log-rank test) showing significantly shorter overall survival in mice injected with TKO SNAhigh cells (p=0.023).
Human subject research was reviewed by Wayne State University IRB and found to not meet the definition of Human Participant Research and therefore exempted from IRB oversight.Samples were collected after obtaining informed consent and deidentified by the Karmanos Cancer Institute Biobanking and Correlative Sciences Core.Omentum samples were consecutively collected from patients undergoing laparoscopic or open surgery for a benign or malignant gynecological condition irrespective of diagnosis or age.

TABLE 1
Differentially regulated pathways in ovarian cancer cells treated with adipose-conditioned media.